Ungulates produced by nuclear transfer of G1 cells

ABSTRACT

A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

This is a continuation of application Ser. No. 09/650,285 filed Aug. 29,2000, which is a continuation of application Ser. No. 08/803,165, filedFeb. 19, 1997 now U.S. Pat. No. 6,252,133, which claims the benefit ofPCT/GB96/02098, filed on Aug. 30, 1996, and British application GB9517779.6, filed on Aug. 31, 1995, all of which are incorporated hereinby reference.

This invention relates to the generation of animals including but notbeing limited to genetically selected and/or modified animals, and tocells useful in their generation.

The reconstruction of mammalian embryos by the transfer of a donornucleus to an enucleated oocyte or one cell zygote allows the productionof genetically identical individuals. This has clear advantages for bothresearch (i.e. as biological controls) and also in commercialapplications (i.e. multiplication of genetically valuable livestock,uniformity of meat products, animal management).

Embryo reconstruction by nuclear transfer was first proposed (Spemann,Embryonic Development and Induction 210-211 Hofner Publishing Co., NewYork (1938)) in order to answer the question of nuclear equivalence or‘do nuclei change during development?’. By transferring nuclei fromincreasingly advanced embryonic stages these experiments were designedto determine at which point nuclei became restricted in theirdevelopmental potential. Due to technical limitations and theunfortunate death of Spemann these studies were not completed until1952, when it was demonstrated in the frog that certain nuclei coulddirect development to a sexually mature adult (Briggs and King, Proc.Natl. Acad. Sci. USA 38 455-461 (1952)). Their findings led to thecurrent concept that equivalent totipotent nuclei from a singleindividual could, when transferred to an enucleated egg, give rise to“genetically identical” individuals. In the true sense of the meaningthese individuals would not be clones as unknown cytoplasmiccontributions in each may vary and also the absence of any chromosomalrearrangements would have to be demonstrated.

Since the demonstration of embryo cloning in amphibians, similartechniques have been applied to mammalian species. These Techniques fallinto two categories: 1) transfer of a donor nucleus to a maturedmetaphase II oocyte which has had its chromosomal DNA removed and 2)transfer of a donor nucleus to a fertilised one cell zygote which hashad both pronuclei removed. In ungulates the former procedure has becomethe method of choice as no development has been reported using thelatter other than when pronuclei are exchanged.

Transfer of the donor nucleus into the oocyte cytoplasm is generallyachieved by inducing cell fusion. In ungulates fusion is induced byapplication of a DC electrical pulse across the contact/fusion plane ofthe couplet. The same pulse which induces cell fusion also activates therecipient oocyte. Following embryo reconstruction further development isdependent on a large number of factors including the ability of thenucleus to direct development i.e. totipotency, developmental competenceof the recipient cytoplast (i.e. oocyte maturation), oocyte activation,embryo culture (reviewed Campbell and Wilmut in Vth World Congress onGenetics as Applied to Livestock 20 180-187 (1994)).

In addition to the above we have shown that maintenance of correctploidy during the first cell cycle of the reconstructed embryo is ofmajor importance (Campbell et al., Biol. Reprod. 49 933-942 (1993);Campbell et al., Biol. Reprod. 50 1385-1393 (1994)). During a singlecell cycle all genomic DNA must be replicated once and only once priorto mitosis. If any of the DNA either fails to replicate or is replicatedmore than once then the ploidy of that nucleus at the time of mitosiswill be incorrect. The mechanisms by which replication is restricted toa single round during each cell cycle are unclear, however, severallines of evidence have implicated that maintenance of an intact nuclearmembrane is crucial to this control. The morphological events whichoccur in the donor nucleus after transfer into an enucleated metaphaseII oocyte have been studied in a number of species including mouse(Czolowiska et al., J. Cell Sci. 69 19-34 (1984)), rabbit (Collas andRobl, Biol. Reprod. 45 455-465 (1991)), pig (Prather et al., J. Exp.Zool. 225 355-358 (1990)), cow (Kanka et al., Mol. Reprod. Dev. 29110-116 (1991)). Immediately upon fusion the donor nuclear envelopebreaks down (NEBD), and the chromosomes prematurely condense (PCC).These effects are catalysed by a cytoplasmic activity termedmaturation/mitosis/meiosis promoting factor (MPF). This activity isfound in all mitotic and meiotic cells reaching a maximal activity atmetaphase. Matured mammalian oocytes are arrested at metaphase of the2nd meiotic division (metaphase II) and have high MPF activity. Uponfertilisation or activation MPF activity declines, the second meioticdivision is completed and the second polar body extruded, the chromatinthen decondenses and pronuclear formation occurs. In nuclear transferembryos reconstructed when MPF levels are high NEBD and PCC occur; theseevents are followed, when MPF activity declines, by chromatindecondensation and nuclear reformation and subsequent DNA replication.In reconstructed embryos correct ploidy can be maintained in one of twoways; firstly by transferring nuclei at a defined cell cycle stage, e.g.diploid nuclei of cells in G1, into metaphase II oocytes at the time ofactivation; or secondly by activating the recipient oocyte andtransferring the donor nucleus after the disappearance of MPF activity.In sheep this latter approach has yielded an increase in the frequencyof development to the blastocyst stage from 21% to 55% of reconstructedembryos when using blastomeres from 16 cell embryos as nuclear donors(Campbell et al., Biol. Reprod. 50 1385-1393 (1994)).

These improvements in the frequency of development of reconstructedembryos have as yet not addressed the question of nuclear reprogramming.During development certain genes become “imprinted” i.e. are alteredsuch that they are no longer transcribed. Studies on imprinting haveshown that this “imprinting” is removed during germ cell formation (i.e.reprogramming). One possibility is that this reprogramming is affectedby exposure of the chromatin to cytoplasmic factors which are present incells undergoing meiosis. This raises the question of how we may mimicthis situation during the reconstruction of embryos by nuclear transferin order to reprogram the developmental clock of the donor nucleus.

It has now been found that nuclear transfer into an oocyte arrested inmetaphase II can give rise to a viable embryo if normal ploidy (i.e.diploidy) is maintained and if the embryo is not activated at the timeof nuclear transfer. The delay in activation allows the nucleus toremain exposed to the recipient cytoplasm.

According to a first aspect of the present invention there is provided amethod of reconstituting an animal embryo, the method comprisingtransferring a diploid nucleus into an oocyte which is arrested in themetaphase of the second meiotic division without concomitantlyactivating the oocyte, keeping the nucleus exposed to the cytoplasm ofthe recipient for a period of time sufficient for the reconstitutedembryo to become capable of giving rise to a live birth and subsequentlyactivating the reconstituted embryo while maintaining correct ploidy. Atthis stage, the reconstituted embryo is a single cell.

In principle, the invention is applicable to all animals, includingbirds such as domestic fowl, amphibian species and fish species. Inpractice, however, it will be to non-human animals, especially non-humanmammals, particularly placental mammals, that the greatest commerciallyuseful applicability is presently envisaged. It is with ungulates,particularly economically important ungulates such as cattle, sheep,goats, water buffalo, camels and pigs that the invention is likely to bemost useful, both as a means for cloning animals and as a means forgenerating transgenic animals. It should also be noted that theinvention is also likely to be applicable to other economicallyimportant animal species such as, for example, horses, llamas orrodents, e.g. rats or mice, or rabbits.

The invention is equally applicable in the production of transgenic, aswell as non-transgenic animals. Transgenic animals may be produced fromgenetically altered donor cells. The overall procedure has a number ofadvantages over conventional procedures for the production of transgenic(i.e. genetically modified) animals which may be summarised as follows:

-   -   (1) fewer recipients will be required;    -   (2) multiple syngeneic founders may be generated using clonal        donor cells;    -   (3) subtle genetic alteration by gene targeting is permitted;    -   (4) all animals produced from embryos prepared by the invention        should transmit the relevant genetic modification through the        germ line as each animal is derived from a single nucleus; in        contrast, production of transgenic animals by pronuclear        injection or chimerism after inclusion of modified stem cell        populations by blastocyst injection produces a proportion of        mosaic animals in which all cells do not contain the        modification and may not transmit the modification through the        germ line; and    -   (5) cells can be selected for the site of genetic modification        (e.g. integration) prior to the generation of the whole animal.

It should be noted that the term “transgenic”, in relation to animals,should not be taken to be limited to referring to animals containing intheir germ line one or more genes from another species, although manytransgenic animals will contain such a gene or genes. Rather, the termrefers more broadly to any animal whose germ line has been the subjectof technical intervention by recombinant DNA technology. So, forexample, an animal in whose germ line an endogenous gene has beendeleted, duplicated, activated or modified is a transgenic animal forthe purposes of this invention as much as an animal to whose germ linean exogenous DNA sequence has been added.

In embodiments of the invention in which the animal is transgenic, thedonor nucleus is genetically modified. The donor nucleus may contain oneor more transgenes and the genetic modification may take place prior tonuclear transfer and embryo reconstitution. Although micro-injection,analogous to injection into the male or female pronucleus of a zygote,may be used as a method of genetic modification, the invention is notlimited to that methodology: mass transformation or transfectiontechniques can also be used e.g. electroporation, viral transfection orlipofection.

In the method of the invention described above, a diploid nucleus istransferred from a donor into the enucleated recipient oocyte. Donorswhich are diploid at the time of transfer are necessary in order tomaintain the correct ploidy of the reconstituted embryo; thereforedonors may be either in the G1 phase or preferably, as is the subject ofour co-pending PCT patent application No. PCT/GB96/02099 filed today(claiming priority from GB 9517780.4), in the G0 phase of the cellcycle.

The mitotic cell cycle has four distinct phases, G, S, G2 and M. Thebeginning event in the cell cycle, called start, takes place in the G1phase and has a unique function. The decision or commitment to undergoanother cell cycle is made at start. Once a cell has passed throughstart, it passes through the remainder of the G1 phase, which is thepre-DNA synthesis phase. The second stage, the S phase, is when DNAsynthesis takes place. This is followed by the G2 phase, which is theperiod between DNA synthesis and mitosis. Mitosis itself occurs at the Mphase. Quiescent cells (which include cells in which quiescence has beeninduced as well as those cells which are naturally quiescent, such ascertain fully differentiated cells) are generally regarded as not beingin any of these four phases of the cycle; they are usually described asbeing in a G0 state, so as to indicate that they would not normallyprogress through the cycle. The nuclei of quiescent G0 cells, like thenuclei of G1 cells, have a diploid DNA content; both of such diploidnuclei can be used in the present invention.

Subject to the above, it is believed that there is no significantlimitation on the cells that can be used in nuclear donors: fully orpartially differentiated cells or undifferentiated cells can be used ascan cells which are cultured in vitro or abstracted ex vivo. The onlylimitation is that the donor cells have normal DNA content and bekaryotypically normal. A preferred source of cells is disclosed in ourco-pending PCT patent application No. PCT/GB95/02095, published as WO96/07732. It is believed that all such normal cells contain all of thegenetic information required for the production of an adult animal. Thepresent invention allows this information to be provided to thedeveloping embryo by altering chromatin structure such that the geneticmaterial can re-direct development.

Recipient cells useful in the invention are enucleated oocytes which arearrested in the metaphase of the second meiotic division. In mostvertebrates, oocyte maturation proceeds in vivo to this fairly latestage of the egg maturation process and then arrests. At ovulation, thearrested oocyte is released from the ovary (and, if fertilisationoccurs, the oocyte is naturally stimulated to complete meiosis). In thepractice of the invention, oocytes can be matured either in vitro or invivo and are collected on appearance of the 1st polar body or as soon aspossible after ovulation, respectively.

It is preferred that the recipient be enucleate. While it has beengenerally assumed that enucleation of recipient oocytes in nucleartransfer procedures is essential, there is no published experimentalconfirmation of this judgement. The original procedure described forungulates involved splitting the cell into two halves, one of which waslikely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)). Thisprocedure has the disadvantage that the other unknown half will stillhave the metaphase apparatus and that the reduction in volume of thecytoplasm is believed to accelerate the pattern of differentiation ofthe new embryo (Eviskov et al., Development 109 322-328 (1990)).

More recently, different procedures have been used in attempts to removethe chromosomes with a minimum of cytoplasm. Aspiration of the firstpolar body and neighbouring cytoplasm was found to remove the metaphaseII apparatus in 67% of sheep oocytes (Smith & Wilmut Biol. Reprod. 401027-1035 (1989)). Only with the use of DNA-specific fluorochrome(Hoechst 33342) was a method provided by which enucleation would beguaranteed with the minimum reduction in cytoplasmic volume (Tsunoda etal., J. Reprod. Fertil. 82 173 (1988)). In livestock species, this isprobably the method of routine use at present (Prather & First J.Reprod. Fertil. Suppl. 41 125 (1990), Westhusin et al., Biol. Reprod.(Suppl.) 42 176 (1990)).

There have been very few reports of non-invasive approaches toenucleation in mammals, whereas in amphibians, irradiation withultraviolet light is used as a routine procedure (Gurdon Q. J. Microsc.Soc. 101 299-311 (1960)). There are no detailed reports of the use ofthis approach in mammals, although during the use of DNA-specificfluorochrome it was noted that exposure of mouse oocytes to ultravioletlight for more than 30 seconds reduced the developmental potential ofthe cell (Tsunoda et al., J. Reprod. Fertil. 82 173 (1988)).

As described above enucleation may be achieved physically, by actualremoval of the nucleus, pro-nuclei or metaphase plate (depending on therecipient cell), or functionally, such as by the application ofultraviolet radiation or another enucleating influence.

After enucleation, the donor nucleus is introduced either by fusion todonor cells under conditions which do not induce oocyte activation or byinjection under non-activating conditions. In order to maintain thecorrect ploidy of the reconstructed embryo the donor nucleus must bediploid (i.e. in the G0 or G1 phase of the cell cycle) at the time offusion.

Once suitable donor and recipient cells have been prepared, it isnecessary for the nucleus of the former to be transferred to the latter.Most conveniently, nuclear transfer is effected by fusion. Activationshould not take place at the time of fusion.

Three established methods which have been used to induce fusion are:

-   -   (1) exposure of cells to fusion-promoting chemicals, such as        polyethylene glycol;    -   (2) the use of inactivated virus, such as Sendai virus; and    -   (3) the use of electrical stimulation.

Exposure of cells to fusion-promoting chemicals such as polyethyleneglycol or other glycols is a routine procedure for the fusion of somaticcells, but it has not been widely used with embryos. As polyethyleneglycol is toxic it is necessary to expose the cells for a minimum periodand the need to be able to remove the chemical quickly may necessitatethe removal of the zona pellucida (Kanka et al., Mol. Reprod. Dev. 29110-116 (1991)). In experiments with mouse embryos, inactivated Sendaivirus provides an efficient means for the fusion of cells fromcleavage-stage embryos (Graham Wistar Inst. Symp. Monogr. 9 19 (1969)),with the additional experimental advantage that activation is notinduced. In ungulates, fusion is commonly achieved by the sameelectrical stimulation that is used to induce parthogenetic activation(Willadsen Nature 320 (6) 63-65 (1986), Prather et al., Biol. Reprod. 37859-866 (1987)). In these species, Sendai virus induces fusion in aproportion of cases, but is not sufficiently reliable for routineapplication (Willadsen Nature 320 (6) 63-65 (1986)).

While cell-cell fusion is a preferred method of effecting nucleartransfer, it is not the only method that can be used. Other suitabletechniques include microinjection (Ritchie and Campbell, J. Reproductionand Fertility Abstract Series No. 15, p60).

In a preferred embodiment of the invention, fusion of the oocytekaryoplast couplet is accomplished in the absence of activation byelectropulsing in 0.3M mannitol solution or 0.27M sucrose solution;alternatively the nucleus may be introduced by injection in a calciumfree medium. The age of the oocytes at the time of fusion/injection andthe absence of calcium ions from the fusion/injection medium preventactivation of the recipient oocyte.

In practice, it is best to enucleate and conduct the transfer s soon aspossible after the oocyte reaches metaphase II. The time that this willbe post onset of maturation (in vitro) or hormone treatment (in vivo)will depend on the species. For cattle or sheep, nuclear transfer shouldpreferably take place within 24 hours; for pigs, within 48 hours; mice,within 12 hours; and rabbits within 20-24 hours. although transfer cantake place later, it becomes progressively more difficult to achieve asthe oocyte ages. High MPF activity is desirable.

Subsequently, the fused reconstructed embryo, which is generallyreturned to the maturation medium, is maintained without being activatedso that the donor nucleus is exposed to the recipient cytoplasm for aperiod of time sufficient to allow the reconstructed embryo to becomecapable, eventually, of giving rise to a live birth (preferably of afertile offspring).

The optimum period of time before activation varies from species tospecies and can readily be determined by experimentation. For cattle, aperiod of from 6 to 20 hours is appropriate. The time period shouldprobably not be less than that which will allow chromosome formation,and it should not be so long either that the couplet activatesspontaneously or, in extreme cases that it dies.

When it is time for activation, any conventional or other suitableactivation protocol can be used. Recent experiments have shown that therequirements for parthogenetic activation are more complicated than hadbeen imagined. It had been assumed that activation is an all-or-nonephenomenon and that the large number of treatments able to induceformation of a pronucleus were all causing “activation”. However,exposure of rabbit oocytes to repeated electrical pulses revealed thatonly selection of an appropriate series of pulses and control of theCa²⁺ was able to promote development of diploidized oocytes tomid-gestation (Ozil Development 109 117-127 (1990)). Duringfertilization there are repeated, transient increases in intracellularcalcium concentration (Cutbertson & Cobbold Nature 316 541-542 (1985))and electrical pulses are believed to cause analogous increases incalcium concentration. There is evidence that the pattern of calciumtransients varies with species and it can be anticipated that theoptimal pattern of electrical pulses will vary in a similar manner. Theinterval between pulses in the rabbit is approximately 4 minutes (OzilDevelopment 109 117-127 (1990)), and in the mouse 10 to 20 minutes(Cutbertson & Cobbold Nature 316 541-542 (1985)), while there arepreliminary observations in the cow that the interval is approximately20 to 30 minutes (Robl et al., in Symposium on Cloning Mammals byNuclear Transplantation (Seidel ed.), Colorado State University, 24-27(1992)). In most published experiments activation was induced with asingle electrical pulse, but new observations suggest that theproportion of reconstituted embryos that develop is increased byexposure to several pulses (Collas & Robl Biol. Reprod. 43 877-884(1990)). In any individual case, routine adjustments may be made tooptimise the number of pulses, the field strength and duration of thepulses and the calcium concentration of the medium.

In the practice of the invention, correct ploidy must be maintainedduring activation. It is desirable to inhibit or stabilise microtubulepolymerisation in order to prevent the production of multiple pronuclei,thereby to maintain correct ploidy. This can be achieved by theapplication of a microtubule inhibitor such as nocodazole at aneffective concentration (such as about 5 μg/ml). Colchecine and colcemidare other microtubule inhibitors. Alternatively, a microtubulestabiliser, such as, for example, taxol could be used.

The molecular component of microtubules (tubulin) is in a state ofdynamic equilibrium between the polymerised and non-polymerised states.Microtubule inhibitors such as nocodazole prevent the addition oftubulin molecules to microtubules, thereby disturbing the equilibriumand leading to microtubule depolymerisation and destruction of thespindle. It is preferred to add the microtubule inhibitor a sufficienttime before activation to ensure complete, or almost complete,depolymerisation of the microtubules. Twenty to thirty minutes is likelyto be sufficient in most cases. A microtubule stabiliser such as taxolprevents the breakdown of the spindle and may also therefore prevent theproduction of multiple pronuclei. Use of a microtubule stabiliser ispreferably under similar conditions to those used for microtubuleinhibitors.

The microtubule inhibitor or stabiliser should remain present afteractivation until pronuclei formation. It should be removed thereafter,and in any event before the first division takes place.

In a preferred embodiment of the invention at 30-42 hours post onset ofmaturation (bovine and ovine, i.e. 6-18 hours post nuclear transfer) thereconstructed oocytes are placed into medium containing nocodazole (5μg/ml) and activated using conventional protocols. Incubation innocodazole may be continued for 4-6 hours following the activationstimulus (dependent upon species and oocyte age).

According to a second aspect of the invention, there is provided aviable reconstituted animal embryo prepared by a method as describedpreviously.

According to a third aspect of the invention, there is provided a methodof preparing an animal, the method comprising:

-   -   (a) reconstituting an animal embryo as described above; and    -   (b) causing an animal to develop to term from the embryo; and    -   (c) optionally, breeding from the animal so formed.

Step (a) has been described in depth above.

The second step, step (b) in the method of this aspect of the inventionis to cause an animal to develop to term from the embryo. This may bedone directly or indirectly. In direct development, the reconstitutedembryo from step (a) is simply allowed to develop without furtherintervention beyond any that may be necessary to allow the developmentto take place. In indirect development, however, the embryo may befurther manipulated before full development takes place. For example,the embryo may be split and the cells clonally expanded, for the purposeof improving yield.

Alternatively or additionally, it may be possible for increased yieldsof viable embryos to be achieved by means of the present invention byclonal expansion of donors and/or if use is made of the process ofserial (nuclear) transfer. A limitation in the presently achieved rateof blastocyst formation may be due to the fact that a majority of theembryos do not “reprogram” (although an acceptable number do). If thisis the case, then the rate may be enhanced as follows. Each embryo thatdoes develop itself can be used as a nuclear donor at the 32-64 cellstage; alternatively, inner cell mass cells can be used at theblastocyst stage. If these embryos do indeed reflect those which havereprogrammed gene expression and those nuclei are in fact reprogrammed(as seems likely), then each developing embryo may be multiplied in thisway by the efficiency of the nuclear transfer process. The degree ofenhancement likely to be achieved depends upon the cell type. In sheep,it is readily possible to obtain 55% blastocyst stage embryos bytransfer of a single blastomere from a 16 cell embryo to a preactivated“Universal Recipient” oocyte. So it is reasonable to hypothesise thateach embryo developed from a single cell could give rise to eight at the16 cell stage. Although these figures are just a rough guide, it isclear that at later developmental stages the extent of benefit woulddepend on the efficiency of the process at that stage.

Aside from the issue of yield-improving expediencies, the reconstitutedembryo may be cultured, in vivo or in vitro to blastocyst.

Experience suggests that embryos derived by nuclear transfer aredifferent from normal embryos and sometimes benefit from or even requireculture conditions in vivo other than those in which embryos are usuallycultured (at least in vivo). The reason for this is not known. Inroutine multiplication of bovine embryos, reconstituted embryos (many ofthem at once) have been cultured in sheep oviducts for 5 to 6 days (asdescribed by Willadsen, In Mammalian Egg Transfer (Adams, E. E., ed.)185 CRC Press, Boca Raton, Fla. (1982)). In the practice of the presentinvention, though, in order to protect the embryo it should preferablybe embedded in a protective medium such as agar before transfer and thendissected from the agar after recovery from the temporary recipient. Thefunction of the protective agar or other medium is twofold: first, itacts as a structural aid for the embryo by holding the zona pellucidatogether; and secondly it acts as barrier to cells of the recipientanimal's immune system. Although this approach increases the proportionof embryos that form blastocysts, there is the disadvantage that anumber of embryos may be lost.

If in vitro conditions are used, those routinely employed in the art arequite acceptable.

At the blastocyst stage, the embryo may be screened for suitability fordevelopment to term. Typically, this will be done where the embryo istransgenic and screening and selection for stable integrants has beencarried out. Screening for non-transgenic genetic markers may also becarried out at this stage. However, because the method of the inventionallows for screening of donors at an earlier stage, that will generallybe preferred.

After screening, if screening has taken place, the blastocyst embryo isallowed to develop to term. This will generally be in vivo. Ifdevelopment up to blastocyst has taken place in vitro, then transferinto the final recipient animal takes place at this stage. If blastocystdevelopment has taken place in vivo, although in principle theblastocyst can be allowed to develop to term in the pre-blastocyst host,in practice the blastocyst will usually be removed from the (temporary)pre-blastocyst recipient and, after dissection from the protectivemedium, will be transferred to the (permanent) post-blastocystrecipient.

In optional step (c) of this aspect of the invention, animals may bebred from the animal prepared by the preceding steps. In this way, ananimal may be used to establish a herd or flock of animals having thedesired genetic characteristic(s).

Animals produced by transfer of nuclei from a source of geneticallyidentical cells share the same nucleus, but are not strictly identicalas they are derived from different oocytes. The significance of thisdifferent origin is not clear, but may affect commercial traits. Recentanalyses of the mitochondrial DNA of dairy cattle in the Iowa StateUniversity Breeding Herd revealed associated with milk and reproductiveperformance (Freeman & Beitz, In Symposium on Cloning Mammals by NuclearTransplantation (Seidel, G. E. Jr., ed.) 17-20, Colorado StateUniversity, Colorado (1992)). It remains to be confirmed that similareffects are present throughout the cattle population and to considerwhether it is possible or necessary in specific situations to considerthe selection of oocytes. In the area of cattle breeding the ability toproduce large numbers of embryos from donors of high genetic merit mayhave considerable potential value in disseminating genetic improvementthrough the national herd. The scale of application will depend upon thecost of each embryo and the proportion of transferred embryos able todevelop to term.

By way of illustration and summary, the following scheme sets out atypical process by which transgenic and non-transgenic animals may beprepared. The process can be regarded as involving five steps:

-   -   (1) isolation of diploid donor cells;    -   (2) optionally, transgenesis, for example by transfection with        suitable constructs, with or without selectable markers;        -   (2a) optionally screen and select for stable integrants—skip            for micro-injection;    -   (3) embryo reconstitution by nuclear transfer;    -   (4) culture, in vivo or in vitro, to blastocyst;        -   (4a) optionally screen and select for stable integrants—omit            if done at 2a—or other desired characteristics;    -   (5) transfer if necessary to final recipient.

This protocol has a number of advantages over previously publishedmethods of nuclear transfer:

1) The chromatin of the donor nucleus can be exposed to the meioticcytoplasm of the recipient oocyte in the absence of activation forappropriate periods of time. This may increase the “reprogramming” ofthe donor nucleus by altering the chromatin structure.

2) Correct ploidy of the reconstructed embryo is maintained when G0/G1nuclei are transferred.

3) Previous studies have shown that activation responsiveness ofbovine/ovine oocytes increases with age. One problem which haspreviously been observed is that in unenucleated aged oocytesduplication of the meiotic spindle pole bodies occurs and multipolarspindles are observed. However, we report that in embryos reconstructedand maintained with high MPF levels although nuclear envelope breakdownand chromatin condensation occur no organised spindle is observed. Theprematurely condensed chromosomes remain in a tight bunch, therefore wecan take advantage of the ageing process and increase the activationresponse of the reconstructed oocyte without adversely affecting theploidy of the reconstructed embryo.

According to a fourth aspect of the invention, there is provided ananimal prepared as described above.

Preferred features of each aspect of the invention are as for each otheraspect, mutatis mutandis.

The invention will now be described by reference to the accompanyingExamples which are provided for the purposes of illustration and are notto be construed as being limiting on the present invention. In thefollowing description, reference is made to the accompanying drawing, inwhich:

FIG. 1 shows the rate of maturation of bovine oocytes in vitro.

EXAMPLE 1 “MAGIC” Procedure Using Bovine Oocytes

Recipient oocytes the subject of this experimental procedure aredesignated MAGIC (Metaphase Arrested G1/G0 AcceptIng Cytoplast)Recipients.

The nuclear and cytoplasmic events during in vitro oocyte maturationwere studied. In addition the roles of fusion and activation in embryosreconstructed at different ages were also investigated. The studies haveshown that oocyte maturation is asynchronous; however, a population ofmatured oocytes can be morphologically selected at 18 hours (FIG. 1).

Morphological Selection of Oocytes

In FIG. 1 ovaries were obtained from a local abattoir and maintained at28-32° C. during transport to the laboratory. Cumulus oocyte complexes(COC's) were aspirated from follicles 3-10 mm in diameter using ahypodermic needle (1.2 mm internal diameter) and placed into sterileplastic universal containers. The universal containers were placed intoa warmed chamber (35° C.) and the follicular material allowed to settlefor 10-15 minutes before pouring off three quarters of the supernatant.The remaining follicular material was diluted with an equal volume ofdissection medium (TCM 199 with Earles salts (Gibco), 75.0 mg/lkanamycin, 30.0 mM Hepes, pH 7.4, osmolarity 280 mOsmols/Kg H₂O)supplemented with 10% bovine serum, transferred into an 85 mm petri dishand searched for COC's under a dissecting microscope.

Complexes with at least 2-3 compact layers of cumulus cells wereselected washed three times in dissection medium and transferred intomaturation medium (TC medium 199 with Earles salts (Gibco), 75 mg/lkanamycin, 30.0 mM Hepes, 7.69 mM NaHCO₃, pH 7.8, osmolarity 280mOsmols/Kg H₂O) supplemented with 10% bovine serum and 1×10⁶ granulosacells/ml and cultured on a rocking table at 39° C. in an atmosphere of5% CO₂ in air. Oocytes were removed from the maturation dish and wetmounted on ethanol cleaned glass slides under coverslips which wereattached using a mixture of 5% petroleum jelly 95% wax. Mounted embryoswere then fixed for 24 hours in freshly prepared methanol: glacialacetic acid (3:1), stained with 45% aceto-orcein (Sigma) and examined byphase contrast and DIC microscopy using a Nikon Microphot-SA, the graphin FIG. 1 shows the percentage of oocytes at MII and those with avisible polar body.

Activation of Bovine Follicular Oocytes

If maturation is then continued until 24 hours these oocytes activate ata very low rate (24%) in mannitol containing calcium (Table 1a).However, removal of calcium and magnesium from the electropulsing mediumprevents any activation.

Table 1a shows activation of bovine follicular oocytes matured in vitrofor different periods. Oocytes were removed from the maturation medium,washed once in activation medium, placed into the activation chamber andgiven a single electrical pulse of 1.25 kV/cm for 80 μs.

TABLE 1a No. Hours post onset of Pronuclear formation of oocytes (N)maturation (hpm) (age hrs)) (% activation) 73 24 24.6 99 30 84.8 55 4592.7* *many 2 or more pronucleiActivation Response of Sham Enucleated Bovine Oocytes

Table 1b shows activation response of in vitro matured bovine oocytessham enucleated at approximately 22 hours post onset of maturation(hpm). Oocytes were treated exactly as for enucleation, a small volumeof cytoplasm was aspirated not containing the metaphase plate. Aftermanipulation the oocytes were given a single DC pulse of 1.25 Kv/cm andreturned to the maturation medium, at 30 hpm and 42 hpm groups ofoocytes were mounted, fixed and stained with aceto-orcein. The resultsshow the number of oocytes at each time point from five individualexperiments as the number of cells having pronuclei with respect to thetotal number of cells.

TABLE 1b No. cells having No. cells having pronuclei/Totalpronuclei/Total no. of cells no. of cells EXPERIMENT 30 hpm 42 hpm 1 1/8— 2 0/24 0/30 3 0/21 0/22 4 0/27 0/25 5 0/19 0/1 hpm = hours post onsetof maturationPronuclear Formation in Enucleated Oocytes

Table 2 shows pronuclear formation in enucleated oocytes fused toprimary bovine fibroblasts (24 hpm) and subsequently activated (42 hpm).The results represent five separate experiments. Oocytes were dividedinto two groups, group A were incubated in nocodazole for 1 hour priorto activation and for 6 hours following activation. Group B were nottreated with nocodazole. Activated oocytes were fixed and stained withaceto-orcein 12 hours post activation. The number of pronuclei (PN) ineach parthenote was then scored under phase contrast. The results areexpressed as the percentage of activated oocytes containing 1 or morepronuclei.

TABLE 2 TOTAL 1 PN 2 PN 3 PN 4 PN >4 PN GROUP A 52 100 0 0 0 0 GROUP B33 45.2 25.8 16.1 3.2 9.7

The absence of an organised spindle and the absence of a polar bodysuggests that in order to maintain ploidy in the reconstructed embryothen only a diploid i.e. G0/G1 nucleus should be transferred into thiscytoplasmic situation. Incubation of activated oocytes in the presenceof the microtubule inhibitor nocodazole for 5 hours, 1 hour prior to andfollowing the activation stimulus prevents the formation of micronuclei(Table 2) and thus when the donor nucleus is in the G0/G1 phase of thecell cycle the correct ploidy of the reconstructed embryo is maintained.

Results

These results show that:

i) these oocytes can be enucleated at 18 hours post onset of maturation(FIG. 1);

ii) enucleated oocytes can be fused to donor blastomeres/cells in either0.3M mannitol or 0.27M sucrose alternatively the donor the cells ornuclei can be injected in calcium free medium in the absence of anyactivation response;

iii) reconstructed embryos or enucleated pulsed oocytes can be culturedin maturation medium and do not undergo spontaneous activation;

iv) the transferred nucleus is seen to undergo nuclear envelopebreakdown (NEBD) and chromosome condensation. No organisedmeiotic/mitotic spindle is observed regardless of the cell cycle stageof the transferred nucleus;

v) such manipulated couplets will activate at 30 hours and 42 hours witha frequency equal to unmanipulated control oocytes;

vi) no polar body is observed following subsequent activation,regardless of the cell cycle stage of the transferred nucleus;

viii) upon subsequent activation 1-5 micronuclei are formed perreconstructed zygote (Table 2).

Reconstruction of Bovine Embryos Using “MAGIC” Procedure

In preliminary experiments this technique has been applied to thereconstruction of bovine embryos using primary fibroblasts synchronisedin the G0 phase of the cell cycle by serum starvation for five days. Theresults are summarised in Table 3.

Table 3 shows development of bovine embryos reconstructed by nucleartransfer of serum starved (G0) bovine primary fibroblasts intoenucleated unactivated MII oocytes. Embryos were reconstructed at 24 hpmand the fused couplets activated at 42 hpm. Fused couplets wereincubated in nocodazole (5 μg/ml) in M2 medium for 1 hour prior toactivation and 5 hours post activation. Couplets were activated with asingle DC pulse of 1.25 KV/cm for 80 μsec.

TABLE 3 NUMBER OF BLASTOCYSTS/ TOTAL NUMBER OF FUSED EXPERIMENT NUMBERCOUPLETS % BLASTOCYSTS 1 1/30 3.3 2 4/31 12.9

EXAMPLE 2 “MAGIC” Procedure Using Ovine Oocytes

Similar observations to those in Example 1 have also been made in ovineoocytes which have been matured in vivo. Freshly ovulated oocytes can beretrieved by flushing from the oviducts of superstimulated ewes 24 hoursafter prostaglandin treatment. The use of calcium magnesium freePBS/1.0% FCS as a flushing medium prevents oocyte activation. Oocytescan be enucleated in calcium free medium and donor cells introduced asabove in the absence of activation. No organised spindle is observed,multiple nuclei are formed upon subsequent activation and this may besuppressed by nocodazole treatment.

Results

In preliminary experiments in sheep, a single pregnancy has resulted inthe birth of a single live lamb. The results are summarised in Tables 4and 5.

Table 4 shows development of ovine embryos reconstructed by transfer ofan embryo derived established cell line to unactivated enucleated invivo matured ovine oocytes. Oocytes were obtained from superstimulatedScottish blackface ewes, the cell line was established from theembryonic disc of a day 9 embryo obtained from a Welsh mountain ewe.Reconstructed embryos were cultured in the ligated oviduct of atemporary recipient ewe for 6 days, recovered and assessed fordevelopment.

TABLE 4 NUMBER OF MORULA, BLA DATE OF STOCYSTS/ NUCLEAR PASSAGE TOTALTRANSFER NUMBER NUMBER 17.1.95 6  4/28 19.1.95 7  1/10 31.1.95 13  0/2 2.2.95 13  0/14  7.2.95 11  1/9  9.2.95 11  1/2 14.2.95 12 16.2.95 13 3/13 TOTAL 10/78 (12.8%)

Table 5 shows induction of pregnancy following transfer of allmorula/blastocyst stage reconstructed embryos to the uterine horn ofsynchronised final recipient blackface ewes. The table shows the totalnumber of embryos from each group transferred the frequency of pregnancyin terms of ewes and embryos, in the majority of cases 2 embryos weretransferred to each ewe. A single twin pregnancy was established whichresulted in the birth of a single live lamb.

TABLE 5 PASSAGE NUMBER “MAGIC” P6 4 P7 1 P11 2 P12 0 P13 3 TOTAL MOR/BL10 TOTAL NUMBER EWES 6 PREGNANT EWES 1 (16.7) % FOETUSES/ 2/10 (20.0)TOTAL TRANSFERRED (%)

1. A method of cloning a pig, comprising: (i) inserting a nucleus of adifferentiated pig cell, which is in the G1 phase of the cell cycle,into an unactivated, enucleated, metaphase II-arrested, pig oocyte, toreconstruct an embryo; (ii) maintaining the reconstructed embryo withoutactivation for a sufficient time to allow the reconstructed embryo tobecome capable of developing to term; (iii) activating the resultantreconstructed embryo; and (iv) transferring said reconstructed embryo toa host pig such that the reconstructed embryo develops into a fetus,wherein the fetus is capable of developing to term.
 2. The methodaccording to claim 1, which further comprises developing the fetus to anoffspring.
 3. The method according to claim 1, wherein saiddifferentiated pig cell is a genetically modified pig cell comprising aninsertion, deletion, or modification.
 4. The method according to claim3, which further comprises developing the fetus to an offspring.
 5. Themethod according to claim 1, which comprises culturing said activatedreconstructed embryo to blastocyst before transferring it to a host. 6.The method according to claim 1, wherein the differentiated pig cell isa fibroblast cell.
 7. The method according to claim 1, wherein thedifferentiated pig cell is from an individual pig that is live-born. 8.The method according to claim 1, wherein the enucleated oocyte ismatured in vitro or in vivo prior to enucleation.
 9. The methodaccording to claim 1, wherein the reconstructed embryo is activated byexposure to several electrical pulses.
 10. The method according to claim1, wherein the reconstructed embryo is activated by exposure to a singleelectrical pulse.
 11. The method according to claim 3, comprising addingan exogenous DNA sequence by microinjection.
 12. The method according toclaim 3, comprising adding an exogenous DNA sequence by electroporation.13. The method according to claim 1, wherein said differentiated pigcell is a cultured pig cell.
 14. A method of cloning a pig, comprising:(i) inserting a nucleus of a cultured, differentiated pig embryonic disccell, which is in the G1 phase of the cell cycle, into an unactivated,enucleated, metaphase II-arrested, pig oocyte, to reconstruct an embryo;(ii) maintaining the reconstructed embryo without activation for asufficient time to allow the reconstructed embryo to become capable ofdeveloping to term; (iii) activating the resultant reconstructed embryo;and (iv) transferring said reconstructed embryo to a host pig such thatthe reconstructed embryo develops into a fetus, wherein the fetus iscapable of developing to term.
 15. The method according to claim 14,which comprises culturing said activated reconstructed embryo toblastocyst before transferring it to a host.
 16. The method according toclaim 14, which further comprises developing the fetus to an offspring.17. A method of producing an ungulate embryo by nuclear transfercomprising: (i) transfer of a nucleus of an ungulate cell, which haspassed start in the mitotic cell cycle and is in the G1 phase of thecell cycle, into an unactivated, enucleated, metaphase II-arrestedungulate oocyte of the same species; (ii) activation of the recipientoocyte containing the donor cell nucleus; and (iii) incubation of theactivated oocyte to provide an embryo; wherein the donor cell nucleus isfrom an ungulate differentiated cell.
 18. The method according to claim17, wherein said ungulate embryo is porcine.